Despite otherwise successful antiretroviral therapy, HIV-associated neurocognitive disorder (HAND) is detected in ~50% of infected individuals. The presence of an HIV-1 reservoir in the brain is considered a contributor to an inflammatory milieu and to the neuronal injury in infected individuals. In the brain, HIV-1 infection is driven mostly by resident microglia cells and infiltrated macrophages. ART is less efficient in the brain tissue because of reduced drug penetration through the blood-brain barrier. Moreover, other than HIV-1 infected T cells that succumb to the cytopathic effect of the virus, microglia/macrophages are resistant to the viral cytopathic effect and can act as long-term producers. Given the long half-life of microglia and the ability of different lineages of macrophages and likely microglia to proliferate, the brain presents itself as a likely reservoir of HIV-1 infection despite ART. In the context of HAND, even if ART were to completely suppress HIV-1 replication, persistently infected microglia/macrophages can actively secrete the HIV-1 transactivator of transcription (Tat) protein, which is known to be particularly toxic for neurons. Tat is known to synergize with drugs of abuse and exacerbate disease progression and severity, further confounding the adverse effects on the central nervous system (CNS). There is mounting evidence that HIV-1 Tat exerts its neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. There is also recent evidence that Tat interacts with amyloid-? plaques, a hallmark of Alzheimer's disease, that are deposited in the brain of HIV-infected patients. Tat secretion thus presents itself as an attractive target to improve HAND. However, our knowledge of how Tat is secreted by infected cells is limited. Conversely, Tat needs to cross membranes of target cells (such as neurons) to exert its neurotoxic effect. Therefore, inhibition of uptake is a second candidate mechanism to improve HAND. Preparation of a stable and functional recombinant full-length Tat has been a barrier to studying the secretion and uptake mechanisms, which also limits the development of drug discovery assays to target the ?active? form of Tat needed to cross membranes. Leading to this application, we have devised new approaches to generate large amounts of stable and functional Tat and employed NMR and biophysical tools to confirm its interaction with membranes. We have also employed a functional assay to confirm Tat?s function in neuronal cell culture. Based on our preliminary data, we hypothesize that Tat undergoes a conformational change upon binding to membranes. Our two specific aims are: (i) identify the molecular rearrangements of Tat upon binding to membrane that facilitate secretion, and (ii) determine the molecular requirements for Tat insertion into endosomal membranes during uptake. The results generated here will provide novel insights into the molecular rearrangements of Tat upon binding to membranes prior to secretion and during uptake and will offer a new platform to target the membrane-bound form of Tat, which altogether will lead to the development of new therapeutic strategies for treatment of HAND.